WO2023075394A1 - Outil d'écouvillon de prélèvement d'échantillon et méthode de détection d'un agent pathogène respiratoire - Google Patents

Outil d'écouvillon de prélèvement d'échantillon et méthode de détection d'un agent pathogène respiratoire Download PDF

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Publication number
WO2023075394A1
WO2023075394A1 PCT/KR2022/016432 KR2022016432W WO2023075394A1 WO 2023075394 A1 WO2023075394 A1 WO 2023075394A1 KR 2022016432 W KR2022016432 W KR 2022016432W WO 2023075394 A1 WO2023075394 A1 WO 2023075394A1
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WIPO (PCT)
Prior art keywords
swab
sample
nucleic acid
tool
gripping point
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PCT/KR2022/016432
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English (en)
Korean (ko)
Inventor
김원식
서명준
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주식회사 씨젠
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Publication of WO2023075394A1 publication Critical patent/WO2023075394A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/08—Detecting, measuring or recording devices for evaluating the respiratory organs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage

Definitions

  • the present invention relates to a sample collection swab tool and a method for detecting respiratory pathogens.
  • Respiratory diseases cause significant mortality, especially in children, the elderly and immunocompromised people. It is very important to identify the causative pathogen for infection control and appropriate patient management. Respiratory pathogen testing has developed rapidly with the development of molecular diagnostic technology, especially real-time PCR. Real-time PCR is more sensitive than traditional methods, and can detect a variety of pathogens simultaneously.
  • UTR upper respiratory tract
  • nasopharyngeal swab specimens and oropharyngeal swab specimens are widely used.
  • sputum and bronchoalveolar lavage (BAL) samples are used as lower respiratory tract samples (Falsey et al., 2012; Branche et al., 2014; Jeong et al., 2014).
  • nasopharyngeal swab samples and oropharyngeal swab samples are used as upper respiratory tract samples, and sputum and bronchial lavage fluid are used as lower respiratory tract samples.
  • the nasopharyngeal swab sample and oropharyngeal swab sample collect secretions from the nasopharynx and oropharynx, respectively, using a cotton swab and store them in a transport medium. and keep
  • nasopharyngeal swab and oropharyngeal swab has limitations that must be performed by experts and is risky as it is an aerosol-generating sampling method.
  • a swab tool for collecting a nasopharyngeal swab sample has a thin thickness and a long length of a fiber layer of a head portion and a support portion adjacent to the head portion in order to collect a sample from the nasopharynx.
  • the swap tool for collecting the oropharyngeal swab sample should be as long as the nasopharyngeal swab tool in order to collect the sample from the oropharynx, but the thickness of the fiber layer of the head portion and the support portion adjacent to the head portion is thicker than that of the nasopharyngeal swab tool.
  • the swap tool for collecting the nasal swab sample may include a stopper that limits the length of the nasal cavity insertion of the head part in order to collect the sample in the nasal cavity, and the length of the swap tool can be shortened for precise force control.
  • the conventional sample collection swap tool has a shape, structure, and/or length that allows a sample to be collected only from a specific sample collection site, so in the case of a kit that collects a sample from two sample collection sites, a sample suitable for a specific sample collection site Must include harvest swap tool.
  • This is not only a limitation for self-sampling by non-experts, but also a problem in economic feasibility because a swap tool having two different shapes, structures, and/or lengths must be included in configuring one package of the sample collection kit. there was.
  • the present inventors have recognized the need to develop a technology capable of customizing and sophisticated power control according to the sample collection site using one swap tool.
  • the present inventors have tried to overcome the above-mentioned problems of the conventional sample collection swap tool and develop a sample collection swap tool that can effectively collect nasal swab samples and oral swab samples.
  • the present inventors formed two gripping positions, that is, two gripping points in one swab tool, and elaborated the swap tool according to the sample collection site (eg, nasal cavity or oral cavity).
  • an object of the present invention is to provide a sample collection swab tool.
  • Another object of the present invention is to provide a sampling kit for detecting respiratory pathogens.
  • Another object of the present invention is to provide a kit for detecting respiratory pathogens.
  • Another object of the present invention is to provide a sampling method for detecting respiratory pathogens.
  • Another object of the present invention is to provide a method for detecting respiratory pathogens.
  • the present invention provides a sampling swab tool comprising:
  • the support portion includes a first gripping point and a second gripping point, and the first gripping point is located adjacent to the head portion.
  • the present inventors have tried to overcome the above-mentioned problems of the conventional sample collection swap tool and develop a sample collection swap tool that can effectively collect nasal swab samples and oral swab samples.
  • the present inventors formed two gripping positions, that is, two gripping points in one swab tool, and it is easy to precisely adjust the force on the swab tool according to the sample collection site (eg, nasal cavity or oral cavity).
  • the sample collection site eg, nasal cavity or oral cavity.
  • Specimens collected using the swab tool of the present invention are respiratory specimens, and specifically, the respiratory specimens are nasal specimens and/or oral specimens.
  • a nasal specimen refers to a biopsy specimen taken from the nasal cavity wall of an empty space in the nose, and is specifically a nostril specimen.
  • An anterior nasal specimen refers to a specimen taken from the nasal wall about 2-3 cm from the front part of the nose, specifically, from the nostril.
  • the present invention is characterized in that it enables self-sampling by non-experts as well as sampling by experts.
  • the swap tool is a self sampling tool.
  • FIGS. 2 to 4 show a sampling swap tool according to one embodiment of the present invention.
  • the sample collection swap tool 100 of the present invention largely includes (a) a head part 10 and (b) a supporting part 11.
  • the head part 10 is a part that directly contacts the sample collection site, and a fiber layer is formed on the head part to collect samples.
  • FIG 5 shows a head portion 10 on which a fiber layer is formed according to one embodiment of the present invention.
  • the fiber layer formed to collect samples in the head part can be formed by various methods known in the art, for example, it can be formed in the head part by a flocking method.
  • adhesive may be applied to the surface of the head portion and small fiber yarns may be vertically deposited in an electrostatic field.
  • the thickness of the fiber layer formed on the head part is 0.2-3 mm.
  • the thickness of the fiber layer is 0.2-3 mm, 0.2-2.8 mm, 0.2-2.5 mm, 0.2-2.2 mm, 0.2-1.8 mm, 0.2-1.5 mm, 0.2-1.2 mm, 0.2-0.8 mm, 0.4-3 0.4-2.8 mm, 0.4-2.5 mm, 0.4-2.2 mm, 0.4-1.8 mm, 0.4-1.5 mm, 0.4-1.2 mm, 0.4-0.8 mm, 0.6-3 mm, 0.6-2.8 mm, 0.6-2.5 mm mm, 0.6-2.2 mm, 0.6-1.8 mm, 0.6-1.5 mm, 0.6-1.2 mm, 0.6-0.8 mm, 1.0-3 mm, 1.0-2.8 mm, 1.0-2.5 mm, 1.0-2.2 mm, 1.0-1.8 mm, 1.0-1.5 mm, 1.0-1.2 mm, 1.2-3 mm, 1.0-2.8 mm, 1.0-
  • the fibers of the fibrous layer may use fibers of various lengths. For example, when forming a fiber layer by a flocking method, fibers of the same length are used to form a uniform thickness.
  • the fiber layer may be formed using fibers having a length of 0.6-3 mm. When the fiber layer is formed using fibers having such a length, the thickness of the fiber layer formed on the head part is 0.6-3 mm.
  • the head portion 10 has an elliptical shape.
  • a fiber layer having a uniform thickness is formed on the elliptical head portion.
  • the head portion on which the fiber layer is formed includes a horizontal long axis portion 101 and a horizontal short axis portion 102.
  • the term "horizontal axis portion” used while referring to the head of the swap tool on which the fiber layer is formed means a portion showing the longest axis length among axes perpendicular to the longitudinal axis of the swap tool in the head of the elliptical swap tool.
  • the "transverse axis portion” means the remaining axis portion except for the portion representing the length of the longest axis among the axes perpendicular to the longitudinal axis.
  • the head portion on which the fiber layer is formed has an oval shape, has a horizontal long axis portion and a horizontal short axis portion, and the thickness of the head portion at the horizontal long axis portion is 3.8 -6.0 mm, and the thickness of the head at the transverse axis is 2.0-3.5 mm.
  • the head portion on which the fiber layer is formed has an oval shape, has a horizontal long axis portion and a horizontal short axis portion, and has a thickness of the head portion at the horizontal long axis portion of 4.8-5.2 mm, The thickness of the head at the transverse axis is 2.8-3.2 mm.
  • the fibers of the fiber layer may have a Dtex value of 3.00-3.50, more specifically, a Dtex value of 3.30-3.35.
  • the above-described thickness and Dtex value of the head portion on which the fibrous layer is formed allow the swab tool to have a suitable absorption capacity and to have a suitable release function when immersed in a specimen transport medium.
  • the fibers of the fibrous layer are materials having water affinity properties, and are specifically selected from the group consisting of polyester, polyamide and cotton.
  • the head unit 10 collects a sample of 30 ⁇ l or more.
  • the amount of the sample to be collected is 30-1500 ⁇ l, 30-1200 ⁇ l, 30-1000 ⁇ l, 30-800 ⁇ l, 30-500 ⁇ l, 30-300 ⁇ l, 30-100 ⁇ l, 50-1500 ⁇ l, 50 ⁇ l.
  • the supporting part 11 is a part that the sampler holds by hand while supporting the sample collection swab tool as a whole, and the force of the sampler is transmitted to the head through the support to collect the sample through the head.
  • the support portion has a cutting groove 12 formed on its outer circumferential surface.
  • the support part 11 may be made of a plastic material.
  • it may be made of polypropylene, polyester or polyamide (PA66 or nylon 66).
  • the support part of the swap tool of the present invention includes two gripping positions, that is, two gripping points.
  • the support part 11 includes a first gripping point 111 and a second gripping point 112 .
  • the first gripping point is located adjacent to the head part.
  • the second gripping point is located closer to the first gripping point than the first gripping point, not adjacent to the head part. Therefore, the second gripping point is located in the order of the head part, the first gripping point, and the second gripping point in the swap tool.
  • the expression “the first gripping point is located adjacent to the head portion” may be expressed as “the first gripping point is located adjacent to the head portion”.
  • tapping point refers to a point or position where a sample collector holds the swab tool by hand.
  • the first gripping point and the second gripping point are each formed as a display unit indicating the gripping point.
  • the display unit is selected from the group consisting of dots, lines, patterns, and grippable shapes.
  • dots, lines, and patterns as a display unit indicating the gripping point
  • dots, lines, and various patterns eg, crosses, stars, triangles, or squares
  • the dots, lines, and patterns may be represented by various colors known in the art (eg, red, blue, or yellow).
  • the first gripping point and the second gripping point are each formed in a grippable shape. More specifically, the first gripping point and the second gripping point are shapes that can be gripped by fingers. More specifically, the grippable shape is a convex shape or a concave shape, and still more specifically a concave shape.
  • 6 and 7 show the shape of the gripping point according to one embodiment of the present invention.
  • a convex shape represents a shape in which curvature is formed outside an axis perpendicular to the longitudinal axis based on the longitudinal axis of the swap tool, and a concave shape forms a curvature inward of an axis perpendicular to the longitudinal axis based on the longitudinal axis of the swap tool. represents a shape.
  • the concave gripping point of the support may be formed in a recessed shape on the outer circumferential surface of the support, and may also be formed in a recessed shape by applying heat and then pressure to the gripping point. there is. More specifically, protrusions may be formed at the gripping points to prevent slipping of the fingers (see FIG. 7).
  • the first gripping point is a gripping position to collect a nasal specimen
  • the second gripping point is a gripping position to collect an oral specimen. More specifically, the first gripping point is a gripping position to collect an anterior nasal cavity specimen, and the second gripping point is a gripping position to collect an oral specimen.
  • the present inventors confirmed that the nasal cavity, among sample collection sites, requires more sophisticated force control than the oral cavity, and that the swab tool should be held shorter when collecting nasal samples than oral samples.
  • the length from the end of the head part to the first gripping point and the length from the end of the head part to the second gripping point have a ratio of 1:1.2-3.
  • the length ratio is 1: 1.2-2.5, 1: 1.2-2.3, 1: 1.2-2.2, 1: 1.2-2.0, 1: 1.4-2.5, 1: 1.4-2.3, 1: 1.4-2.2, 1:1.4-2.0, 1:1.5-2.5, 1:1.5-2.3, 1:1.5-2.2 or 1:1.5-2.0.
  • the end of the head part indicates an end of the head part that is not adjacent to the support part.
  • first gripping point and the second gripping point have the above ratio of lengths based on the end of the head part, precise force control is possible so that samples can be collected from two or more sampling sites through one swab tool.
  • the positions of the first gripping point and the second gripping point adopted in the present invention may be determined based on the cutting groove formed in the support part.
  • the first gripping point is located between the head part and the cutting groove
  • the second gripping point is located between the cutting groove and an end of the support part.
  • the end of the support part indicates the end of the support part that is not adjacent to the head part.
  • the swap tool of this implementation can be seen in FIG. 2 .
  • the swap tool of this embodiment is composed of a head part, a first gripping point, a cutting groove, and a second gripping point in order.
  • the length from the end of the head part to the first gripping point and the length from the end of the head part to the second gripping point have a ratio of 1:1.8-2.5.
  • the end of the head part refers to the end of the head part that is not adjacent to the support part.
  • the length ratios are 1: 1.8-2.5, 1: 1.8-2.3, 1: 1.8-2.2, 1: 1.8-2.0, 1: 1.9-2.5, 1: 1.9-2.3, 1: 1.9-2.2, 1 : 1.9-2.0, 1 : 2.0-2.5, 1 : 2.0-2.3 or 1 : 2.0-2.2.
  • first gripping point and the second gripping point have the above ratio of lengths based on the end of the head part, precise force control is possible so that samples can be collected from two or more sampling sites through one swab tool.
  • the first gripping point and the second gripping point are located between the cutting groove and the end of the support part.
  • the end of the support part indicates the end of the support part that is not adjacent to the head part.
  • the swap tool of this implementation can be seen in FIG. 3 .
  • the swap tool of this embodiment is composed of a head part, a cutting groove, a first gripping point, and a second gripping point in this order.
  • the length from the end of the head part to the first gripping point and the length from the end of the head part to the second gripping point have a ratio of 1:1.2-1.8.
  • the end of the head part refers to the end of the head part that is not adjacent to the support part.
  • the length ratios are 1: 1.2-1.8, 1: 1.2-1.7, 1: 1.2-1.6, 1: 1.2-1.5, 1: 1.3-1.8, 1: 1.3-1.7, 1: 1.3-1.6, 1:1.3-1.5, 1:1.4-1.8, 1:1.4-1.7, 1:1.4-1.6, 1:1.4-1.5, 1:1.5-1.8, 1:1.5-1.7 or 1:1.5-1.6 .
  • first gripping point and the second gripping point have the above ratio of lengths based on the end of the head part, precise force control is possible so that samples can be collected from two or more sampling sites through one swab tool.
  • FIGS. 8 is a process of collecting nasal and oral swab samples using the sample collection swap tool of FIGS. 2 and 3, cutting the swap tool at a cutting groove, dipping the swab sample into a sample transport medium, and transporting the swab sample to a test institution indicates Specifically, in the case of collecting a nasal swab sample using the sample collection swap tool of FIGS. 2 and 3, hold the swap tool briefly using the first gripping point, insert the swab tool into the nose and gently scrape to collect the sample, and then collect the sample using the swap tool. into a tube containing a sample transport medium, cut the swab tool in the cutting groove, seal the cut head by dipping, and transport it to a testing institution.
  • the support portion additionally includes a cutting groove formed on an outer circumferential surface thereof.
  • the positions of the first gripping point and the second gripping point adopted in the present invention may be determined based on two cutting grooves formed in the support part.
  • the cutting groove of the support part is a first cutting groove
  • the first cutting groove is located between the head part and the first gripping point
  • the support part additionally includes a second cutting groove.
  • the second cutting groove is located between the first gripping point and the second gripping point.
  • the swap tool of this implementation can be seen in FIG. 4 .
  • the swap tool of this embodiment is composed of a head part, a first cutting groove, a first gripping point, a second cutting groove, and a second gripping point in this order.
  • FIG. 9 illustrates a process of collecting nasal and oral swab samples using the sample collection swap tool of FIG. 4, cutting the swab tool at the cutting groove, dipping the swab sample into a sample transport medium, and transporting the swab sample to a test institution. .
  • the sample collection swap tool of FIG. 4 cutting the swab tool at the cutting groove, dipping the swab sample into a sample transport medium, and transporting the swab sample to a test institution.
  • the swab tool is cut at the first cut groove, and the cut head is sealed by dipping and transported to an inspection agency.
  • the swap tool is cut at the second cutting groove, and the cut head is sealed by dipping and transported to an inspection agency.
  • the length from the end of the head part to the first gripping point and the length from the end of the head part to the second gripping point have a ratio of 1:1.1-1.6.
  • the end of the head part refers to the end of the head part that is not adjacent to the support part.
  • the length ratio is 1: 1.1-1.6, 1: 1.1-1.5, 1: 1.1-1.4, 1: 1.1-1.3, 1: 1.1-1.2, 1: 1.15-1.6, 1: 1.15-1.5, 1:1.15-1.4, 1:1.15-1.3 or 1:1.15-1.2.
  • first gripping point and the second gripping point have the above ratio of lengths based on the end of the head part, precise force control is possible so that samples can be collected from two or more sampling sites through one swab tool.
  • the length from the end of the head part to the first cutting groove and the length from the end of the head part to the first gripping point have a ratio of 1:1.5-2.5.
  • the end of the head part refers to the end of the head part that is not adjacent to the support part.
  • the length ratios are 1: 1.5-2.5, 1: 1.5-2.3, 1: 1.5-2.1, 1: 1.5-1.9, 1: 1.7-2.5, 1: 1.7-2.3, 1: 1.7-2.1, 1:1.7-1.9, 1:1.8-2.5, 1:1.8-2.3, 1:1.8-2.1 or 1:1.8-1.9.
  • the length from the end of the head part to the second cutting groove and the length from the end of the head part to the second gripping point have a ratio of 1:1.2-1.8.
  • the end of the head part refers to the end of the head part that is not adjacent to the support part.
  • the length ratios are 1: 1.2-1.8, 1: 1.2-1.7, 1: 1.2-1.6, 1: 1.2-1.5, 1: 1.3-1.8, 1: 1.3-1.7, 1: 1.3-1.6, 1:1.3-1.5, 1:1.4-1.8, 1:1.4-1.7, 1:1.4-1.6, 1:1.4-1.5, 1:1.5-1.8, 1:1.5-1.7 or 1:1.5-1.6 .
  • the length from the end of the head part to the first cutting groove and the length from the end of the head part to the second cutting groove have a ratio of 1:1.8-2.5.
  • the end of the head part refers to the end of the head part that is not adjacent to the support part.
  • the length ratios are 1: 1.8-2.5, 1: 1.8-2.4, 1: 1.8-2.3, 1: 2.0-2.5, 1: 2.0-2.4, 1: 2.0-2.3, 1: 2.2-2.5, It may be 1 : 2.2-2.4 or 1 : 2.2-2.3.
  • the head part and the support part have a length ratio of 1:5-15.
  • the length ratio is 1:5-15, 1:5-13, 1:5-11, 1:5-9, 1:7-15, 1:7-13, 1:7-11, It may be 1:7-9, 1:9-15, 1:9-13 or 1:9-11.
  • the overall length of the swap tool is 140-180 mm.
  • the overall length of the swap tool 100 is 140-180 mm, 140-176 mm, 140-172 mm, 140-168 mm, 140-164 mm, 140-160 mm, 144-180 mm, 144- 176 mm, 144-172 mm, 144-168 mm, 144-164 mm, 144-160 mm, 148-180 mm, 148-176 mm, 148-172 mm, 148-168 mm, 148-164 mm, 148- 160 mm, 152-180 mm, 152-176 mm, 152-172 mm, 152-168 mm, 152-164 mm, 152-160 mm, 156-180 mm, 156-176 mm, 156-172 mm, 156- 168 mm, 156-164 mm, 156-160 mm, 160-180 mm, 160-176 mm, 160-172 mm, 160-172 mm, 160-180 mm
  • the length of the head portion is 14-18 mm.
  • the length of the head portion 10 is 14-18 mm, 14-17 mm, 14-16 mm, 15-18 mm, 15-17 mm, 15-16 mm, 16-18 mm or 16-17 mm can be
  • the sample can be collected by directly contacting the sample collection site.
  • the length of the support is 120-160 mm.
  • the length of the support part 11 is 120-160 mm, 120-156 mm, 120-152 mm, 120-148 mm, 120-144 mm, 124-160 mm, 124-156 mm, 124-152 mm , 124-148 mm, 124-144 mm, 128-160 mm, 128-156 mm, 128-152 mm, 128-148 mm, 128-144 mm, 132-160 mm, 132-156 mm, 132-152 mm , 132-148 mm, 132-144 mm, 136-160 mm, 136-156 mm, 136-152 mm, 136-148 mm, 136-144 mm, 140-160 mm, 140-156 mm, 140-152 mm , 140-148 mm, 140-144 mm, 144-160 mm, 144-156 mm, 140-152 mm , 140-148 mm, 140-144
  • the sample collection swab tool may be supported as a whole.
  • the swap tool is a swap tool for collecting a respiratory sample, specifically a swap tool for collecting a nasal and oral sample, and more specifically a swap tool for collecting an anterior nasal cavity and oral sample.
  • a sampling kit for detecting respiratory pathogens comprising:
  • sample collection kit of the present invention includes the above-described sample collection swap tool of the present invention, descriptions of common contents between the two are omitted in order to avoid excessive complexity in the present specification.
  • the sample collection kit is a self-sampling kit.
  • the specimen transport medium is a cell preservation medium or a cell inactivation medium.
  • the cell preservation medium is a saline-based solution or a balanced salt solution-based medium.
  • the saline-based solution is phosphate buffered saline (PBS) or normal saline.
  • the cell inactivation medium contains (i) a chaotropic agent. More specifically, the cell inactivation medium is (ii) detergent; (iii) chelators; (iv) a buffer; and (v) a reducing agent.
  • the sample transport medium when the sample transport medium is impregnated with the sample collected by the sample collection swap tool, it is used for a nucleic acid amplification reaction without a nucleic acid separation process.
  • the sample transport medium further contains a pH indicator (eg, phenol red, bromocresol purple, and bromothymol blue).
  • a pH indicator eg, phenol red, bromocresol purple, and bromothymol blue.
  • the present invention provides a kit for detection of a respiratory pathogen comprising:
  • a real-time nucleic acid amplification reaction composition comprising (i) a primer for amplifying the nucleic acid molecule of the respiratory pathogen, (ii) a probe hybridized to the nucleic acid molecule of the respiratory pathogen, and (iii) an enzyme including DNA polymerase .
  • kit for detecting respiratory pathogens of the present invention includes the above-described sample collection kit of the present invention, descriptions of common contents between the two are omitted in order to avoid excessive complexity in the present specification.
  • the enzymes include hot start Taq polymerase and reverse transcriptase.
  • the nucleic acid amplification reaction composition further includes primers for amplifying endogenous IC and exogenous IC.
  • the endogenous IC is RNase P gene, betaglobin gene, GAPDH gene, beta actin gene, PPIA gene, RPS9 gene, RPS15A gene or UXT gene, and the exogenous IC is bacteriophage MS2 or Amourd (armoured) RNA.
  • the respiratory pathogen is a respiratory virus and/or a respiratory bacterium. More specifically, the respiratory virus is influenza virus, respiratory syncytial virus (RSV), adenovirus, enterovirus, parainfluenza virus (PIV), metapneumovirus (MPV), bocavirus, rhinovirus and / or coronavirus. More specifically, the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • the nucleic acid molecule of the respiratory pathogen is at least one of the RdRP gene, S gene and N gene of SARS-CoV-2.
  • the present invention provides a sampling method for detecting respiratory pathogens comprising the steps of:
  • the sample collection method of the present invention uses the above-described sample collection kit of the present invention, and descriptions of common details between the two are omitted in order to avoid excessive complexity in the present specification.
  • the present invention provides a method for detecting respiratory pathogens comprising the following steps:
  • a nucleic acid amplification reaction is performed using a transport medium and a nucleic acid amplification reaction composition in which the nasal swab sample and the oral swab sample collected by the above-described sample collection swab tool of the present invention are dipped together. carrying out;
  • the method for detecting respiratory pathogens of the present invention uses the above-described sample collection swap tool of the present invention, and descriptions of common contents between the two are omitted in order to avoid excessive complexity in the present specification.
  • the detection method of the present invention is described according to each step as follows:
  • a nucleic acid amplification reaction composition and a transport medium in which the nasal swab sample and the oral swab sample collected by the sample collection and swap tool of the present invention are dipped together are used. Carry out an amplification reaction.
  • the specimen used in the present invention is a respiratory swab specimen, specifically a nasal swab specimen and an oral swab specimen, and more specifically, a total nasal swab specimen and an oral swab specimen.
  • nasal swab specimen refers to a swab specimen in which a biopsy in the nasal cavity obtained by applying a swab tool to the nasal cavity is adsorbed, and is a specimen distinct from a nasopharyngeal swab specimen.
  • the nasal cavity is the anterior nasal cavity.
  • a swab tool is inserted into the nasal cavity (eg, inserted 2-3 cm), and a biopsy is taken from the inner portion of the nasal cavity while gently turning the swab for 10-15 seconds. .
  • nasal swab specimens are obtained by applying one swab tool to both nasal cavities.
  • the oral swab specimen used in the present invention may include a specimen obtained by applying a swab tool to the oral cavity.
  • oral swab specimen refers to a specimen obtained by collecting saliva and/or epithelial cells of the oral wall by applying it to the oral cavity using a swab tool.
  • oral swab sample refers to a sample obtained by collecting saliva by applying a swab tool to the oral cavity (ie, saliva swab sample).
  • the oral swab sample is obtained by applying the swab tool to the lingual part, sublingual part or oral wall.
  • the swab tool is applied to the surface of the tongue so that the fibrous layer of the swab tool is sufficiently absorbed, or the saliva is collected from the tip of the tongue and then the fibrous layer of the swab tool is sufficiently absorbed (see FIG. 11),
  • An oral swab sample can be obtained by rubbing the swab tool on the oral wall so that saliva is sufficiently absorbed into the fibrous layer and epithelial cells are adsorbed to the fibrous layer.
  • an oral swab sample may be obtained by swabbing the sublingual portion so that saliva is sufficiently absorbed (see FIG. 11).
  • the oral swab specimen is obtained by applying the swab tool to the sublingual region.
  • the oral swab sample can be obtained by applying the swab tool to the inside of the cheek, gum, and/or palate.
  • the saliva swab sample may be used in combination with an oral swab sample or an oral saliva sample.
  • the nasal swab specimen and the oral swab specimen are obtained by applying the two swab tools respectively to the nasal cavity and the oral cavity.
  • nasal swabs and oral swabs are obtained by applying one swab tool to the nasal cavity and then to the oral cavity.
  • nasal swab specimens and oral swab specimens are obtained through self-sampling.
  • Nasal swab specimens and oral swab specimens are specimens that can be easily performed and obtained by ordinary people without the involvement of experts, and therefore, effective specimens necessary for molecular diagnosis can be obtained even by self-sampling.
  • sample collection swap tool for collecting the nasal swab sample and the oral swab sample uses the above-described sample collection swap tool of the present invention, the description of the common content between the two is omitted in order to avoid excessive complexity in the present specification. .
  • the sampled nasal swab and oral swab are dipped, ie, impregnated, into the sample transport medium in the container. By this dipping, the sample contained in the swab is released into the sample transport medium.
  • a container containing a sample transport medium into which a swab is dipped is transported to a place where a nucleic acid amplification reaction is performed.
  • sample transport medium described in the method of the present invention is equally applicable to the sample transport medium included in the sample collection kit of the present invention described above.
  • the specimen transport medium that can be used in the present invention includes two mediums: a cell maintenance buffer and a cell inactivation buffer (i.e., cell disruption medium).
  • cell preservation medium refers to a medium that maintains cells in a specimen so as not to be disrupted.
  • the cell preservation medium that can be used in the present invention includes any cell preservation medium commonly used in the art.
  • the cell preservation medium is a saline-based solution or a balanced salt solution-based medium.
  • the saline-based solution used in the present invention is phosphate buffered saline (PBS) or normal saline.
  • PBS phosphate buffered saline
  • normal saline normal saline
  • the balanced salt solution-based medium used in the present invention may include a balanced salt solution commonly used in the art.
  • balanced salt solutions include Alsever's solution, Earle's balanced salt solution, Gey's balanced salt solution, Hanks' balanced salt solution, These include Puck's balanced salt solution, Ringer's balanced salt solution, Simm's balanced salt solution and Tyrode's balanced salt solution.
  • the balanced salt solution-based medium used in the present invention may further include other components in the above-described balanced salt solution.
  • the other components include bovine serum albumin, cysteine, sucrose and glutamic acid.
  • the cell inactivation medium used in the present invention i.e., disruptive medium, disrupts a specimen, such as a virus, to release nucleic acid molecules within the virus into the medium, denatures protein components, inactivates DNase and RNase, and Integrity is a medium to maintain.
  • the cell inert medium as the sample transport medium used in the present invention contains (i) a chaotropic agent, and more specifically (i) in addition to the chaotropic agent (ii) dieter detergent; (iii) chelators; (iv) a buffer; and (v) at least one component selected from the group consisting of a reducing agent, more specifically, in addition to (i) a chaotropic agent (ii) a detergent; (iii) chelators; (iv) a buffer; and (v) a reducing agent.
  • the chaotropic agent is guanidine thiocyanate, guanidine isocyanate, guanidine hydrochloride and potassium thiocyanate, more specifically, guanidine thiocyanate.
  • Chaotropic agents open microbial cells, induce cell lysis, release DNA and RNA, and inhibit degradation of nucleic acid molecules by nucleases.
  • the detergent is sodium dodecyl sulfate, lithium dodecyl sulfate, sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate, sodium deoxycholate, sodium cholate, sodium alkylbenzene sulfonate, polysorbate, Triton X-100 or N-lauroyl sarcosine.
  • the chelator is ethylene glycol tetraacetic acid, hydroxyethylethylenediaminetriacetic acid, diethylene triamine pentaacetic acid, N,N-bis(carboxymethyl)glycine, ethylenediaminetetraacetic, citrate anhydrous, sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate, citric acid, diammonium citrate, ferric ammonium citrate or lithium citrate.
  • the buffer is tris (hydroxymethyl) aminomethane, citrate, 2- (N-morpholino) ethanesulfonic acid, N, N-Bis (2-hydroxyethyl) -2-aminoethanesulfonic acid, 1,3-bis (tris (hydroxymethyl) methyl amino)propane, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, 3-(N-morpholino)propanesulfonic acid, hydroxyethyl piperazine ethane sulfonic acid, bicarbonate and phosphate.
  • the reducing agent is 2-mercaptoethanol, tris (2-carboxyethyl) phosphine, dithiothreitol, dimethylsulfoxide and sodium hydroxide.
  • the chaotropic agent may be included in an amount of 5-30 parts by weight based on 100 parts by weight of the medium, the detergent is 1-15 parts by weight, the chelator is 0.1-5 parts by weight, and the buffer solution 2-10 parts by weight of silver and 0.1-3 parts by weight of the reducing agent may be included.
  • the cell inactivation medium as a sample transport medium has an inactivating function by disruption of respiratory infectious pathogens and nucleic acid materials (specifically, DNA or RNA, more specifically RNA) released from the disrupted pathogens. has a stabilizing function.
  • the sample transport medium further includes a pH indicator (eg, phenol red, bromocresol purple, and bromothymol blue), more specifically, phenol red.
  • a pH indicator eg, phenol red, bromocresol purple, and bromothymol blue
  • the pH indicator makes it possible to monitor whether the sample transport medium is infected with bacteria.
  • a nucleic acid amplification reaction is carried out using a sample transport medium.
  • Sample transport media can be applied to nucleic acid amplification reactions in three ways:
  • the specimen transport medium is directly applied to the nucleic acid amplification reaction without any pretreatment.
  • PCR can be performed by adding a sample transport medium to a nucleic acid amplification reaction composition according to a conventional direct PCR method.
  • the incubation result is applied to the nucleic acid amplification reaction.
  • the sample transport medium in which the nasal swab sample and the oral swab sample are dipped together is incubated at 60°C to 100°C (more specifically, 95°C to 100°C) for 1 minute to 15 minutes before being used in the nucleic acid amplification reaction. (incubation) treatment, and the nucleic acid amplification reaction is performed using the result of the incubation.
  • the specimen transport medium used in the nucleic acid amplification reaction may be diluted 2 to 10 times (more specifically, 3 to 5 times) before or after incubation and applied to the nucleic acid amplification reaction.
  • a nucleic acid separation process is applied to the sample transport medium in which the nasal swab sample and the oral swab sample are dipped together before being used for the nucleic acid amplification reaction, and the nucleic acid molecules separated in the nucleic acid separation process are used. Carry out an amplification reaction.
  • the nucleic acid separation process may be performed, for example, according to a magnetic particle-based separation method (see US Patent Nos. 6027945 and 7517969).
  • nucleic acid molecules can be separated by sequentially using a buffer for separating nucleic acid molecules composed of a lysis buffer, a binding buffer, a washing buffer, and an elution buffer.
  • the sample transport medium is treated with a lysis buffer containing a chaotropic agent (e.g., guanidine thiocyanate) and a detergent (e.g., Tween-20) to disrupt viruses or bacteria in the sample, and magnetic particles are added to the disruption product.
  • a chaotropic agent e.g., guanidine thiocyanate
  • a detergent e.g., Tween-20
  • Nucleic acid molecules bound to the surface of the magnetic particles are separated by treatment with a binding buffer containing , washing the magnetic particles with a washing buffer containing ethanol, and then treatment with an elution buffer to separate the nucleic acid molecules bound to the surfaces of the magnetic particles.
  • primers and probes that hybridize to the target may be used.
  • the probe or primer used in the present invention has a sequence complementary to the target nucleotide sequence.
  • the term “complementary” means that it has sufficient complementarity to selectively hybridize to the above-described nucleotide sequence under certain specific hybridization or annealing conditions. Therefore, the term “complementary” has a different meaning from the term perfectly complementary, and the primers or probes of the present invention have one or more mismatches ( mismatch) base sequence.
  • primer refers to a single molecule capable of acting as a starting point for template-directed DNA synthesis under suitable conditions (i.e., four different nucleoside triphosphates and polymerases) in a suitable buffer at a suitable temperature. -means a stranded oligonucleotide.
  • suitable conditions i.e., four different nucleoside triphosphates and polymerases
  • the suitable length of a primer varies depending on various factors, such as temperature and use of the primer, but is typically 15-40 nucleotides. Shorter primer molecules generally require lower temperatures to form a sufficiently stable hybrid complex with the template.
  • the sequence of the primer does not have to have a sequence completely complementary to a part of the sequence of the template, and it is sufficient to have sufficient complementarity within the range of hybridizing with the template to perform the specific function of the primer. Therefore, the primer in the present invention does not have to have a sequence perfectly complementary to the above-described nucleotide sequence as a template, and it is sufficient to have sufficient complementarity within the range in which it can hybridize to this gene sequence and act as a primer.
  • the design of such primers can be easily performed by those skilled in the art by referring to the above-described nucleotide sequences, and can be performed using, for example, a primer design program (eg, PRIMER 3 program).
  • probe refers to a natural or modified monomer or linear oligomer of linkages, including deoxyribonucleotides and ribonucleotides, capable of specifically hybridizing to a target nucleotide sequence, and naturally existing or artificially synthesized.
  • the probe of the present invention is preferably single-stranded and is an oligodeoxyribonucleotide.
  • amplification refers to a reaction that amplifies a nucleic acid molecule.
  • PCR polymerase chain reaction
  • RT-PCR reverse transcription-polymerase chain reaction
  • nucleic acid sequence-based amplification based amplification (NASBA), US Pat. Nos. 5,130,238, 5,409,818, 5,554,517, and 6,063,603), strand displacement amplification (21, 22) and loop-mediated isothermal amplification; LAMP) 23, but is not limited thereto.
  • Other amplification methods that may be used are described in U.S. Patent Nos. 5,242,794, 5,494,810, 4,988,617 and U.S. Patent No. 09/854,317.
  • PCR is the most well-known nucleic acid amplification method, and many variations and applications thereof have been developed. For example, touchdown PCR, hot start PCR, nested PCR and booster PCR have been developed by modifying traditional PCR procedures to enhance the specificity or sensitivity of PCR.
  • real-time PCR differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), multiplex PCR, inverse polymerase chain reaction chain reaction (IPCR), vectorette PCR and thermal asymmetric interlaced PCR (TAIL-PCR) have been developed for specific applications.
  • DD-PCR differential display PCR
  • RACE rapid amplification of cDNA ends
  • IPCR inverse polymerase chain reaction chain reaction
  • TAIL-PCR thermal asymmetric interlaced PCR
  • RNA Ribonucleic acid
  • a gene amplification reaction is performed using RNA in the sample as a template and primers binding to RNA or cDNA.
  • cDNA is synthesized from the isolated RNA, and this cDNA is amplified.
  • cDNA can be readily synthesized using reverse transcriptase (PNAS USA, 85:8998 (1988); Libert F, et al., Science , 244:569 (1989); and Sambrook, J. et al. , Molecular Cloning.A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)). Subsequently, the synthesized cDNA is amplified through a gene amplification reaction.
  • the primer used in the present invention is hybridized or annealed to one site of the template to form a double-stranded structure.
  • Conditions of nucleic acid hybridization suitable for forming such a double-stranded structure are described in Joseph Sambrook, et al., Molecular Cloning , A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization , A Practical Approach, IRL Press, Washington, DC (1985).
  • DNA polymerases can be used for the amplification of the present invention, including the “Klenow” fragment of E. coli DNA polymerase I, thermostable DNA polymerase and bacteriophage T7 DNA polymerase.
  • the polymerase is a thermostable DNA polymerase obtained from various bacterial species, which include Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis , Thermis flavus , Thermococcus literalis , and Pyrococcus furiosus (Pfu).
  • Thermus aquaticus Taq
  • Thermus thermophilus Tth
  • Thermus filiformis Thermis flavus
  • Thermococcus literalis Thermococcus literalis
  • Pyrococcus furiosus Pfu
  • the reaction vessel When carrying out the polymerization reaction, it is preferable to provide the reaction vessel with components necessary for the reaction in excess.
  • the excess of components required for the amplification reaction means an amount to the extent that the amplification reaction is not substantially limited to the concentration of the components. It is required to provide cofactors such as Mg 2+ , dATP, dCTP, dGTP and dTTP to the reaction mixture in such a way that the desired degree of amplification can be achieved.
  • All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, the buffer allows all enzymes to approach optimal reaction conditions. Thus, the amplification process of the present invention can be carried out in a single reactant without changing conditions such as addition of reactants.
  • the nucleic acid amplification reaction used in the present invention is a real-time nucleic acid amplification reaction.
  • the real-time nucleic acid amplification reaction may be performed using a non-specific fluorescent dye that is non-specifically intercalated with a duplex, which is an amplicon of a target nucleic acid sequence.
  • the real-time nucleic acid amplification reaction may use a labeled probe that specifically hybridizes to a target nucleic acid sequence.
  • the method is a molecular beacon method (Tyagi et al, Nature Biotechnology v.14 MARCH 1996) using a dual-labeled probe that forms a hairpin structure, as a donor or acceptor.
  • Hybridization probe method using two single labeled probes (Bernad et al, 147-148 Clin Chem 2000; 46) and Lux method using single labeled oligonucleotides (U.S. Patent No. 7,537,886), dual labeled
  • the TaqMan method U.S. Patent Nos. 5,210,015 and 5,538,848, which utilizes hybridization of probes as well as cleavage of dual-labeled probes by 5'-nuclease activity of DNA polymerase.
  • a real-time nucleic acid amplification reaction can be performed using a dimer formed depending on the presence of a target nucleic acid sequence.
  • the dimer formed depending on the presence of the target nucleic acid sequence is not an amplification product of the target sequence formed by the amplification reaction itself, but is a dimer whose amount increases in proportion to the amplification of the target nucleic acid sequence.
  • a dimer formed depending on the presence of a target nucleic acid sequence can be obtained by various methods, for example, by the PTO Cleavage and Extension (PTOCE) method disclosed in WO 2012/096523, the above patent documents being incorporated herein by reference. inserted into
  • the nucleic acid amplification reaction composition includes a primer, a probe and an enzyme.
  • the nucleic acid amplification reaction composition includes a buffer, dNTP, KCl and MgCl 2 .
  • the nucleic acid amplification reaction composition may include 40 mM Tris ⁇ Cl (pH 8.8), 100 mM KCl, 4 mM MgCl 2 and 400 ⁇ M dNTP.
  • Enzymes included in the nucleic acid amplification reaction composition of the present invention include Taq DNA polymerase (specifically, hot start Taq DNA polymerase) and reverse transcriptase, and more specifically, hot start Taq DNA polymerase, reverse transcriptase, UDG (Uracil DNA glycosylase) and RI (RNase inhibitor).
  • the hot start Taq DNA polymerase is chemically modified hot start Taq DNA polymerase (Reference: Paul N, et al., Hot start PCR. Methods in Molecular Biology. Humana Press. 630:301-18 (2010)), antibody- based hot start Taq DNA polymerase (Paul N, et al., Hot start PCR. Methods in Molecular Biology. Humana Press.
  • the reverse transcriptase used in the present invention is a reverse transcriptase commonly used in the art, specifically an MMLV-based thermostable reverse transcriptase.
  • the nucleic acid amplification reaction composition includes primers, probes and enzymes (hot start Taq DNA polymerase, MMLV-based thermostable reverse transcriptase and UDG).
  • an internal control is used in a nucleic acid amplification reaction to determine the validity of the nucleic acid amplification reaction.
  • the sampling effectiveness of the nasal swab specimen and oral swab specimen as well as the nucleic acid amplification reaction can be determined using two ICs.
  • the nucleic acid amplification reaction composition includes primers for amplifying nucleic acid molecules of the respiratory pathogen, endogenous IC and exogenous IC, wherein the endogenous IC is The sampling validity of nasal swabs and oral swabs is determined and the exogenous IC is used to determine the validity of the nucleic acid amplification reaction.
  • the endogenous IC is RNase P gene, betaglobin gene, GAPDH gene, beta actin gene, PPIA gene, RPS9 gene, RPS15A gene or UXT gene, and the exogenous IC is bacteriophage MS2 or Amourd (armoured) RNA.
  • the presence or absence of the respiratory pathogen is determined by analyzing the result of the nucleic acid amplification reaction.
  • a signal of a real-time nucleic acid amplification reaction is detected in real time, and when a signal equal to or higher than a specific threshold value is observed, it is determined that a respiratory pathogen is present.
  • the respiratory pathogens detected by the present invention are respiratory viruses and/or respiratory bacteria.
  • influenza virus eg, influenza A virus and influenza B virus
  • respiratory syncytial virus eg RSV A and RSV B
  • adenovirus enterovirus
  • parainfluenza virus eg , PIV 1, PIV 2, PIV 3 and PIV 4
  • MPV metapneumovirus
  • bocavirus eg CoV NL63, CoV 229E, CoV OC43, CoV HKU1, SARS-CoV, MERS-CoV, SARS-CoV-2
  • Mycoplasma pneumoniae Chlamydophila pneumoniae , Legionella pneumophila , Haemophilus influenzae , Streptococcus pneumoniae , Bordetella pertussis and/or Bordetella parapertussis infection.
  • the present invention is an influenza virus, RSV (respiratory syncytial virus), adenovirus, enterovirus, PIV (parainfluenza virus), MPV (metapneumovirus), bocavirus, rhinovirus and / or coronavirus infection by respiratory viruses, including
  • the method of the present invention determines infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • the nucleic acid amplification reaction composition is used for amplification of at least one, more specifically at least two, more specifically three genes among the RdRP gene, S gene and N gene of SARS-CoV-2 It is determined that the pathogen of SARS-CoV-2 is present when at least one of the RdRP gene, the S gene, and the N gene is measured as positive.
  • the present invention is performed by a real-time nucleic acid amplification reaction that exhibits a LoD (Limit of Detection) value of 100-500 RNA copies/reaction.
  • LoD Light of Detection
  • a conventional sample collection swap tool has a shape, structure, and/or length that allows a sample to be collected only from a specific sampling site, so a kit that collects a sample from two sampling sites is suitable for a specific sampling site.
  • a sample collection swap tool should be included. This is not only a limitation for self-sampling by non-experts, but also a problem in economic feasibility because a swap tool having two different shapes, structures, and/or lengths must be included in configuring one package of the sample collection kit. there was.
  • the present invention forms two positions that can be gripped in one swab tool, that is, two gripping points, and a precise force on the swab tool according to the sample collection site (eg, nasal cavity or oral cavity) By facilitating control, it is possible to accurately and easily collect a swab sample from the sample collection site and collect a sufficient amount of sample to detect respiratory pathogens.
  • the sample collection site eg, nasal cavity or oral cavity
  • the swap tool of the present invention can be used not only for sampling by experts but also for self-sampling by non-experts, and can detect respiratory pathogens from collected samples.
  • FIG. 1 shows a conventional sampling swap tool having different shapes, structures, and/or lengths depending on the sampling site.
  • FIG. 5 shows a head portion of a sample collection swab tool according to an embodiment of the present invention.
  • 6 and 7 show the shape of the gripping point according to one embodiment of the present invention.
  • FIGS. 8 illustrates a process of collecting nasal and oral swab samples using the sample collection swab tool of FIGS. 2 and 3, cutting the swab sample at a cutting groove, dipping the swab sample into a sample transport medium, and transporting the swab sample to a test institution.
  • FIG. 9 illustrates a process of collecting nasal and oral swab samples using the sample collection swap tool of FIG. 4 , cutting the swab sample at a cutting groove, dipping the swab sample into a sample transport medium, and transporting the swab sample to a test institution.
  • the nasal swab sample collected using the swab tool of FIG. 4 is cut at the first cutting groove and then dipped into the sample transport medium, and the oral swab sample collected using the swab tool of FIG. 4 is cut at the second cutting groove. It is then dipped into the sample transport medium.
  • 10 exemplarily shows how to collect a sample from the anterior nasal cavity.
  • 11 exemplarily shows how to collect a sample from the oral cavity.
  • Comparative Example 1 Detecting SARS-CoV-2 by taking a nasal swab sample and an oral swab sample
  • Nasopharyngeal and oropharyngeal swab samples were collected using the nasopharyngeal swab sample and oropharyngeal swap sample collection swap tool (Copan Company) included in 1 package of the sample collection kit among the sample collection swap tools shown in FIG. 1 .
  • nasopharyngeal swab specimen and the oropharyngeal swab specimen were impregnated in a balanced salt solution-based medium, CTM (Clinical Virus Transport Medium) (Noble Bio Company) or UTM (Copan Company) specimen transport medium.
  • CTM Circal Virus Transport Medium
  • UTM Copan Company
  • the target nucleic acid was obtained by heat treatment by diluting the nasopharyngeal swab specimen and the specimen transport medium impregnated with the oropharyngeal swab specimen 3 times and incubating at 98° C. for 3 minutes (hereinafter, referred to as “crude nucleic acid”).
  • Nucleic acid isolation was performed using Seegene's STARMag 96X4 Universal Cartridge Kit (Cat. No. 744300.4.UC384, Seegene Inc.) and automatic nucleic acid extraction equipment Microlab NIMBUS (Cat. No. 65415-02, Hamilton). Nucleic acid separation was carried out according to the manufacturer of the nucleic acid separation reagent and the device operation manual. Nucleic acid isolation was performed using 300 ⁇ l of the specimen transport medium impregnated with the nasopharyngeal swab specimen and the oropharyngeal swab specimen (hereinafter referred to as "isolated nucleic acid").
  • Allplex TM SARS-CoV-2 Assay Seegene
  • Allplex TM SARS-CoV-2 Assay product is a one-step RT-PCR product for detecting Sarbecovirus and SARS-CoV-2 target gene E gene, SARS-CoV-2 target gene RdRP gene, S gene and N gene.
  • the tubes each containing the prepared reaction mixture were placed in a real-time thermocycler (CFX96, Bio-Rad) and reacted at 50 ° C for 20 minutes, then denatured at 95 ° C for 15 minutes, 95 ° C for 10 seconds, 60 ° C 15 sec at 72 ° C. and 10 sec were repeated for 45 cycles. The above experiment was repeated twice to obtain an average Ct value.
  • CFX96 real-time thermocycler
  • Example 1 Detecting SARS-CoV-2 by taking an anterior nasal swab sample and an oral swab sample
  • the sampling swap tool of FIG. 3 has an overall length of 160 mm, a head portion length of 16 mm, and a support portion length of 144 mm. And, in the swap tool of FIG. 3, the lengths from the end of the head not adjacent to the support to the cutting groove, the length to the first gripping point, and the length to the second gripping point are 60 mm, 80 mm, and 120 mm, respectively. am.
  • the sample collection swab tool of FIG. 3 includes an elliptical head on which a fiber layer is formed, and the head has a thickness of 5.0 mm in the transverse axis and 3.0 mm in the transverse axis.
  • the fiber layer of the swab is formed by coating polyester fibers of 3.30-3.35 Dtex with flocking technology.
  • the first gripping point of the swab tool was gripped, the swab tool was inserted 2 cm into the nasal cavity, and swabbing was performed while gently rotating for 20 seconds to obtain an anterior nasal swab sample. Swabing was performed in one anterior nasal cavity, followed by swabbing in the other anterior nasal cavity. Subsequently, the second gripping point of the swab tool was gripped, and the swab tool was applied to the sublingual area to swab so that saliva was sufficiently absorbed into the fiber layer of the swab to obtain an oral swab sample.
  • the swab tool obtained with the nasal swab sample is cut at the first cutting groove
  • the swab tool obtained with the oral swab sample is cut at the second cutting groove
  • Specimens and oral swab samples were impregnated in a balanced salt solution-based medium, CTM (Clinical Virus Transport Medium) (Noble Bio Company) or UTM (Copan Company) specimen transport medium.
  • target nucleic acids to be added to the RT-PCR reaction were obtained by heat-treating the specimen transport medium or through a nucleic acid isolation process.
  • the target nucleic acid was obtained by heat treatment by diluting the specimen transport medium impregnated with the nasal swab specimen and the oral swab specimen 3 times and incubating at 98° C. for 3 minutes (hereinafter, referred to as “crude nucleic acid”).
  • Nucleic acid isolation was performed using Seegene's STARMag 96X4 Universal Cartridge Kit (Cat. No. 744300.4.UC384, Seegene Inc.) and automatic nucleic acid extraction equipment Microlab NIMBUS (Cat. No. 65415-02, Hamilton). Nucleic acid separation was carried out according to the manufacturer of the nucleic acid separation reagent and the device operation manual. Nucleic acid isolation was performed using 300 ⁇ l of the specimen transport medium impregnated with the whole nasal swab specimen and the oral swab specimen (hereinafter referred to as "isolated nucleic acid").
  • Allplex TM SARS-CoV-2 Assay Seegene
  • Allplex TM SARS-CoV-2 Assay product is a one-step RT-PCR product for detecting Sarbecovirus and SARS-CoV-2 target gene E gene, SARS-CoV-2 target gene RdRP gene, S gene and N gene.
  • the tubes each containing the prepared reaction mixture were placed in a real-time thermocycler (CFX96, Bio-Rad) and reacted at 50 ° C for 20 minutes, then denatured at 95 ° C for 15 minutes, 95 ° C for 10 seconds, 60 ° C 15 sec at 72 ° C. and 10 sec were repeated for 45 cycles. The above experiment was repeated twice to obtain an average Ct value.
  • CFX96 real-time thermocycler

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Abstract

La présente invention concerne un outil d'écouvillon de prélèvement d'échantillon et une méthode de détection d'agents pathogènes respiratoires. La présente invention concerne un outil d'écouvillon dans lequel deux positions pour saisir l'outil d'écouvillon, c'est-à-dire deux points de préhension, sont formées de manière à faciliter le contrôle précis de la force sur l'outil d'écouvillon selon le site de prélèvement d'échantillon (par exemple, une cavité nasale ou une cavité buccale), moyennant quoi des échantillons d'écouvillon peuvent être prélevés à partir de sites de prélèvement d'échantillon avec précision et facilement en une quantité suffisante pour détecter des agents pathogènes respiratoires. De plus, l'outil d'écouvillon selon la présente invention peut être utilisé non seulement pour un prélèvement par des experts mais également pour un auto-prélèvement par des non-experts, et des agents pathogènes respiratoires peuvent être détectés à partir d'échantillons prélevés.
PCT/KR2022/016432 2021-10-29 2022-10-26 Outil d'écouvillon de prélèvement d'échantillon et méthode de détection d'un agent pathogène respiratoire WO2023075394A1 (fr)

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